Method of concentrating and recovering a viral enzyme activity from biological samples

ABSTRACT

A method of concentrating and recovering an enzyme activity from enveloped viruses present in a biological sample, is described. The method comprises contacting the biological sample in a first buffer solution with a virus-binding matrix, such as an anion exchanger matrix, to attach virus particles present in the sample to the matrix, washing the matrix carrying the virus particles with a second buffer solution to remove components interfering with viral enzyme activity, lysing the immobilized virus particles in a third buffer solution and recovering the concentrated viral enzyme activity from the third buffer solution. Additionally, a commercial package containing written and/or data carrier instructions for performing laboratory steps for concentration and recovery of an enzyme activity from enveloped viruses present in a biological sample and at least one component necessary for the assay, is disclosed.

This application is a national stage filing under 35 U.S.C. 371 ofPCT/SE01/00617, filed Mar. 22, 2001.

The present invention relates to a method of concentrating andrecovering a viral enzyme activity from biological samples. Moreprecisely, the invention relates to a method of concentrating andrecovering an enzyme activity from enveloped viruses present in abiological sample. The invention also relates to a commercial packageuseful in the performance of the method of the invention.

BACKGROUND OF THE INVENTION

In a virion (virus particle), the viral nucleic acid is covered by aprotein capsid, and in some of the more complex animal viruses, thecapsid itself is surrounded by an envelope containing membrane lipidsand glycoproteins. Among these enveloped viruses, the ones receivingmost attention today are the different retroviruses, especially HIV(Human immunodeficiency virus). The name retrovirus comes from thedirection of the flow of genetic information for these viruses, from RNAto the DNA of the cell. Some retroviral strains are found to besymbiotic in one species and pathogenic in another. These findingsindicate a potentially severe problem with xenotransplantation(transplantation of animal organs in humans). Both endogenous andexogenous retroviruses can contribute to vertical and horizontaltransmission of genetic material within and between species and providemechanisms for evolution of new pathogenic agents.

During the last decade there has been an increasing demand for methodsfor diagnosis and monitoring of retroviral infections as well as forresearch on the pathogenesis of new retroviruses. The state of the artis best illustrated by the methods currently used in the management ofHIV infection. In the laboratory HIV-infection is detected and monitoredby measurement of a) antibodies to HIV antigen, b) circulating HIVantigen, c) virus isolation, d) HIV RNA in circulation, and e) provirusDNA integrated in cells. The method of choice depends on the purpose ofthe test, and the stage of disease.

The currently accepted marker for viral load in the clinical setting ismeasurement of copy number of HIV RNA in plasma This can be accomplishedeither by PCR, NASBA, or by branched DNA techniques [Revets el al.,1996]. Currently used assays have a detection limit of 20 RNA copies/mlplasma [Perrin et al., 1998]. As all techniques for detection of viralnucleic acids are based on the binding of sequence specific primers,there is a significant risk that they will not hybridize to new strainsor sequence variants in a sample.

Another important aspect concerning in vitro measurements of infectionparameters emerges when extrapolating the results for the in vivosituation. Several of the laboratory techniques involve an amplificationstage during which a selection step occurs. In the worst case the majorviral variants amplified are those best matching the primers used, notthe most abundant in the in vivo situation.

Direct measurement of viral enzymes in samples from the patient's bloodwould therefore be an attractive alternative to cell culture or thenucleic acid based methods both for quantification of viral replicationand for studies of the effect of antiviral substances. The obstacles toovercome are both the assay sensitivity and to reproducibly separate theenzyme from host-specific enzymes and activity inhibiting substances.

The present invention enables measurement of an enzyme activity from anenveloped virus present in a biological sample by concentrating andrecovering the enzyme activity without isolating the virions.

The prior art methods designed for concentration and purification ofdifferent viruses are either specific methods based on the antigenicproperties of the virus proteins or generalized methods based on basicchemical or physical characteristics of the virus. Only the latter typeof methods can be considered relevant for purification of antigenicallyhighly variable retrovirus like HIV.

Viruses have traditionally been partly purified by different types ofcentrifugation, either by just pelleting the virus or in combinationwith the use of precipitating agents such as polyethylene glycol (U.S.Pat. No. 5,661,022). Centrifugation in density gradients results in morepure virus preparations (U.S. Pat. No. 4,729,955).

Another approach has been to bind the virus to different types ofadsorbents. Porath and Jansson (U.S. Pat. No. 3,925,152) used insolubleorganic macroporous polymers selected from the group consisting of agar,agarose, dextran and cellulose containing amphoteric substitutentscomposed of both basic nitrogen containing groups and carboxylate orsulphonated groups: Adsorbed viruses could in their system be elutedwith solutions of successively different ionic strength to obtain virusvariant separation. Their pioneering work has during the last twodecades been followed in many related purification techniques based oninteraction of viruses with gels with either anionic or cationicionexchange residues (U.S. Pat. No. 3,655,509). Retroviruses and therelated hepatitis B virus has for example been purified using bothcation exchange (U.S. Pat. No. 5,447,859, U.S. Pat. No. 4,138,287) oranion exchange (U.S. Pat. No. 5,837,520, U.S. Pat. No. 5,661,023). Themethods cited above are all based on binding the virus at low ionicstrength and elution at high ionic strength.

Yet another approach for purification of virus and viral nucleic acid isbased on binding virus to a water insoluble cross-linked polyhydroxypolycarboxylic acid polymer (U.S. Pat. No. 5,658,779). The virus isbound to the polymer at pH 6.0 to 8.0, probably by hydrophobicinteractions and can be desorbed at pH 8.0 to 11.0. One interestingapplication of this technique concerns isolation of viral nucleic acid.In one such example bacteriophages from a bacterial lysate are in afirst step adsorbed to the polymer. The polymer is then washed and thebound virus is disrupted using either a chelating agent, like EDTA, or adenaturating agent, like SDS. The viral proteins then adsorb to thepolymer and a viral nucleic acid essentially free of interactingproteins is achieved. Use of a similar approach for purification ofviral proteins is claimed but the technique described will release theviral proteins together with all other cellular and viral componentsadsorbed to the polymer.

The present inventors have conducted extensive research on measurementsof the activity of the viral enzyme reverse transcriptase (RT). RTactivity has previously been measured directly in serum or plasma [Awad,R. J. K. et al 1997, Ekstrand et al 1996]. One problem encountered inthese early studies was the induction of high titers of RT activityinhibiting antibodies soon after primary infection. Another was theoccasional occurrence of RT inhibitors in samples from HIV negativecontrols.

DESCRIPTION OF THE INVENTION

The present invention provides a solution to the above mentioned priorart problems. Further, it can be used for concentration of viral enzymeactivity from large volumes of fluids i.e. cell culture supernatantscontaining very small virus amounts. Another application is to transferviral enzyme from virions in highly inhibitory samples such as extractsfrom tissues or cultured cells into an environment that is compatiblewith enzyme assay conditions.

Thus, the current invention provides a method of concentrating andrecovering viral enzyme activity, particularly from enveloped viruses,from crude biological samples such as blood, plasma, serum, cell culturefluid and cell extracts. It is based on a separation step in which thevirions are captured on a solid matrix. Enzyme inhibitory antibodies andother inhibitory substances are removed by a wash step and the enzymesare released by lysing the immobilized virions. Both the binding and thelysis steps are critical. The binding step must have the capacity toalmost quantitatively immobilize small amounts of virions frombiological fluids containing very high protein concentrations, such asserum or plasma, preferentially without binding immunoglobulin (Ig) orother potentially inhibitory substances. The lysis step should nearlyquantitatively release the viral enzymes into an environment whichprevents the enzyme from binding to the separation matrix, stabilizesthe native enzyme and is compatible with optimal assay conditions.

In particular, the present invention is directed to a method ofconcentrating and recovering an enzyme activity from enveloped virusespresent in a biological sample comprising the steps of

contacting the biological sample in a first buffer solution at aconcentration in range of 100-500 mM of buffering substance and having apH of 4.0-8.5 and optionally containing chaothropic ions at an ionicstrength of up to 2 M with a virus-binding matrix, to attach virusparticles present in the sample to the matrix,washing the matrix carrying the virus particles with a second buffersolution at a concentration of 1-100 mM of buffering substance andcontaining cations at a concentration of 0.1-1 M and having a pH of 4-9,to remove components interfering with viral enzyme activity,lysing the immobilized virus particles in a third buffer solution at aconcentration of 10-500 mM of buffering substance and containing a nondenaturing detergent and having a pH of 6-9, andrecovering the concentrated viral enzyme activity from the third buffersolution.

In an embodiment of the invention the virus-binding matrix is an anionexchanger matrix, e.g. an anion exchanger matrix containing tertiaryand/or quaternary amine groups, such as Fractogel® EMD DMAE andFractogel® EMD TMAE equilibrated in 150 mM MES(2-(N-Morpholino)ethanesulfonic acid) pH 6.0.

In further embodiments the first buffer is selected from the groupconsisting of 150 mM MES pH 6.0, and 200 mM Potassium iodide (KI), thesecond buffer is selected from the group consisting of 10 mM MES pH 6.0,and 500 mM Potassium acetate (KAc), and the third buffer is selectedfrom the group consisting of an enzyme assay compatible buffer includinga detergent, e.g. 1% Triton X 100 and a buffering substance, e.g. 100 mMN-(2-Hydroxyethylpiperazine-N′-(2-ethanesulfonic acid) (Hepes) pH 7.6.

In a particularly preferred embodiment the concentrated and recoveredviral enzyme activity is reverse transcriptase (RT) activity.

The biological sample is preferably selected from the group consistingof serum and plasma samples.

The present invention is also directed to a commercial package useful inthe performance of the method of the invention. The package containswritten and/or data carrier instructions for performing laboratory stepsfor concentration and recovery of an enzyme activity from envelopedviruses present in a biological sample, and

at least one of the components

a virus-binding matrix,

a first buffer solution at a concentration in range of 100-500 mM ofbuffering substance and having a pH of 4.0-8.5 and containingchaothropic ions at an ionic strength of up to 2 M,

a second buffer solution at a concentration of 1-100 mM of bufferingsubstance and containing cations at a concentration of 0.1-1 M andhaving a pH of 4-9,

a third buffer solution at a concentration of 10-500 mM of bufferingsubstance and containing a non denaturing detergent and having a pH of4-9, Mini columns, and

Plastic tubes.

In a preferred embodiment of the commercial package the virus-bindingmatrix is an anion exchanger matrix,

the first buffer is selected from the group consisting of 150 mM MES pH6.0, and 200 mM Potassium iodide (KI),

the second buffer is selected from the group consisting of 10 mM MES pH6.0, and 500 mM Potassium acetate (KAc), and

the third buffer is selected from the group consisting of a RT assaycompatible buffer including a detergent, e.g. 1% Triton X 100 and abuffering substance, e.g. 100 mM(N-(2-Hydroxyethylpiperazine-N′-(2-ethanesulfonic acid) (Hepes) pH 7.6.

In a particularly preferred embodiment the anion exchanger matrix is ananion exchanger matrix containing tertiary and/or quaternary aminegroups, such as Fractogel® EMD DMAE and Fractogel® EMD TMAE equilibratedin 150 mM MES (2-(N-Morpholino)-ethanesulfonic acid) pH 6.0.

Considerations Regarding Choice of Buffers

A) Binding Buffer (the First Buffer)

The binding buffer is used to adjust the pH and ionic strength in thesample to values giving maximal binding of the virus particles and atthe same time minimizing binding of host components, such asplasma/serum proteins, and particularly antibodies as well as otherpotentially inhibitory substances.

1) pH

With an anion exchanger and FIV virus we found binding with buffersbetween pH 7.5 and 5.0 in a system without plasma components. Withregard to the isoelectric point (Ip) of the plasma components whichshould not bind, a pH as low as possible should be used. Fresh EDTAplasma samples has a pH of approximately 8.5. Depending on handling(e.g. storage) the pH in such samples may drop to approximately 8.0.

A number of buffers were investigated with regard to the ability tonormalize pH in different plasmas to a defined pH in the range 3.0-7.5.

Two problems were encountered with pH values below 6.0.

-   a) pH values below 5.0 were not possible to achieve with    concentrations of buffer up to 0.5 M.-   b) When using a 1:1 mixture of plasma and buffer, precipitates    occurred at pH values below 6.0. Centrifugation experiments    confirmed that virus particles in plasma also started to precipitate    at pH below 6.0

Chosen values for the preferred pH range is therefore 4.0-8.5, and pH6.0 is considered optimal.

The chosen concentration of buffer should be in the range of 100-500 mM,and 150 mM is considered optimal.

Two buffers functioning in the pH range of approximately 6.0 wereinvestigated, namely(bis(2-Hydroxyethyl)iminotris(hydroxymethyl)methane;2-bis(2-Hydroxyethyl)-amino-2(hydroxymethyl)-1,3-propanediol) (Bis-Tris)and MES.

Bis-Tris is basic and requires pH adjustment with large amounts of acid.At different pH values a mixture of pH effect and ionic strength istherefore obtained MES is acidic and requires less adjustment to givethe required pH. Binding experiments indicated that the MES bufferfunctions slightly better, and this buffer was selected for studies onthe significance of the ionic strength.

II) Ionic Strength

As the pH range was decided upon, we investigated at which ionicstrength and with which negative ions present the virus particles stillbound. Sulfate, acetate, chloride and iodide were primarily used.

Two ions far apart in the chaothropic series, acetate (Ac) and iodide,were studied in a broader concentration range. The effects were theexpected: The iodide started to affect the binding of virus at a lowerconcentration than acetate.

The binding was reduced with 50% by 500 mM of iodide, but 2 M of Ac isrequired for this effect.

The amount of bound Ig that could be eluted from the gel wasinvestigated after binding and washing of FIV in plasma with differentcombinations of Ac and iodide in the buffers.

Having no salts present at the binding gave higher residual amounts ofIg after lysis (even though the gel had already been washed with salt).Iodide was more efficient than Ac, that gave approximately 1.5 timeshigher amount of residual Ig. By combining different salts in the twosteps it was shown that iodide in the binding but Ac in the wash wasequally efficient as iodide throughout.

Useful negative ions may be weak eluents like Ac to strong eluents asiodide as well as those ions found between these with regard toproperties in the chaothropic series, preferably Ac, Cl and iodide, andparticularly iodide.

Chosen values for the ionic strength may be varied and may be up to 2 M,preferably 0.1-1 M, and particularly 200 mM for iodide and 1 M for Ac,respectively.

B) Wash Buffer (the Second Buffer)

1) The buffer shall, as effectively as possible, remove factors whichmay interfere with the measurement/recovery of viral enzymes from thegel. It is particularly important to remove antibodies.

2) The buffer must not remove virions from the gel or lyse virions.

3) Since the components of the buffer, after a certain dilution (approx.5-9 times), will be present in the enzyme assay, the components may notseverely inhibit the enzyme.

Critical factors for the wash buffer are the pH and the ionic strength.

I) pH

Proteins bind and remain bound to an anion exchanger at pH above theirisoelectric point. The Ip of different immunoglobulins varies, but lieson the average at pH 8.9.

Our experiments with FIV virions showed that virus in a buffercontaining 0.1 M KCI did bind very well to several different anionexchangers in the pH range of 5.8-7.6. The recovery at lysis afterwashing with buffer at the same pH was high throughout the whole pHrange. Ip for FIV is thus<5.8.

The chosen pH range for the wash buffer is pH 4-9 (even though possiblylower pH values may be possible to use).

Preferably the pH range is 5.5-8 (the washing effect on Ig is likely tobe poor above 8.0), and especially 6-7.5. The buffer substance should bepresent at a relatively low concentration which is sufficient to keepthe pH stable, but which does not require a too high concentration ofbuffer substance to change the pH at the lysis stage.

Chosen range for the concentration of buffer substance is 1-100 mM.Preferably 5-50 mM and optimally 10 mM.

II) Ionic Strength

Too high concentrations of cation may release virus at wash, too lowconcentrations give residual Ig and other inhibiting substances in thelysate.

The concentration limits are influenced by the actually used caeothropiccation. The principal is to use as high a concentration as possiblewhich does not release the virus.

The concentration is also limited by the effect of the ion in the enzyme(e.g. RT) assay after dilution (approx. 5-9 times), see Example 4.

Different anionic exchangers probably give more or less residual Ig,depending on the properties of the matrix, at a given concentration ofsalt.

For example, the following applies when Fractogel TMAE is washed at pH6.0.

1) Wash with Ac up to 0.5 M no loss of virus. At 1 M recovery of approx.50%. Up to 0.25 M still approx. 0.2% residual Ig. At 0.5 M andhigher<0.02% residual Ig.

2) Wash with chloride (Cl⁻) up to 0.25 M no loss of virus, at 0.5 Mapprox. 50%, at 1 M all the virus is lost. Residual Ig at Cl⁻ of 0.25 Mand higher<0.02%.

3) High concentration of iodine in the absence of serum protein seems toirreversibly affect the virus so that we could not recover any enzyme(RT) activity. Iodine is therefore good at the binding stage but shouldnot be used in the wash buffer.

Chosen range for the concentration of cation in wash buffer is 0.1-1 M,preferably 0.2-0.6 M, and particularly Ac⁻ or Cl⁻ is used at 0.2-0.6 M.

C) Lysis Buffer (the Third Buffer)

The lysis step should nearly quantitatively release the viral enzymesinto an environment which prevents the enzyme from binding to theseparation matrix, stabilizes the native enzyme and is compatible withoptimal assay conditions.

I) Detergent

Detergent is necessary for the lysis of the virus. There are a vastnumber of detergents, and in the present context the detergent should bea “non denaturating detergent” e.g. Polyoxyethanesorbitan (Tween) ort-Octylphenoxypolyethoxyethanol (Triton X-100) Different detergents aremore or less effective for lysis of virions and their interference inthe enzyme (RT) assay at high concentrations is rather minimal.Therefore no specific range for the concentration is necessary. It maybe mentioned that Triton X 100 is not effective at 0.1% and starts toinduce problems at 5%.

II) pH

The pH must be compatible with the pH of the chosen enzyme assay.Furthermore, the pH shall not allow binding of viral enzyme to the ionexchanger. We have found that there is no binding of FIV or HIV RT at pH5.5-8.5, and that Ip for HIV RT lies in the vicinity of 9.1. The effectof pH on enzyme assay is therefore of major importance.

Chosen pH range is 4-9, preferably 7-8, and optimally 7.6.

The buffering substance shall be added at a sufficiently highconcentration to adjust the pH from 6.0 to 7.6. The requiredconcentration varies of course with the concentration in the washbuffer. Too high a concentration may interfere in the enzyme assay.Chosen range of the concentration of buffering substance is 10-500 mM,preferably 50-200 mM, and optimally 100 mM.

Of course the buffer solution may contain additional componentsfavorable for the chosen enzyme assay.

The invention will now be illustrated by the following description ofembodiments, particularly procedures adapted to provide RT samples,which can be accurately analyzed with regard to RT activity by our(Cavidi Tech) commercial colorimetric RT assays, e.g. described in ourSwedish patent application 9902410-1. These embodiments and examples arenot intended to limit the scope of the claimed invention.

General Description of the Performance of the Method of the Invention

Two slightly different embodiments of the invention are described.

I) Protocol for Batch Separation of Plasma.

-   1) Label the desired number of 15 ml plastic centrifuge tubes to    identify the samples to be analyzed.-   2) Suspend the separation gel carefully into a 1:1 gel slurry, and    transfer 400 μl gel slurry to each labeled centrifuge tube. Spin    down the gel and remove the buffer.-   3) Mix 500 μl of each plasma to be analysed with an equal volume of    the binding buffer (A).

Transfer each plasma-buffer mixture to the corresponding labeledcentrifuge tube with gel. Add stopper, shake the tubes to suspend thegel into the sample and incubate for one hour on an orbital shaker atroom temperature.

-   4) Prepare the wash buffer (B), 30 ml buffer/sample is needed-   5) Allow the gel to settle and remove the supernatant. Add 10 ml    wash buffer (B). Put the stopper back, resuspend the gel into the    wash buffer by shaking. Let the gel sediment for approximately 10    minutes. Remove stopper, aspirate the wash fluid carefully to avoid    aspirating gel.-   6) Repeat the wash according to 6.-   7) Repeat the wash according to 6.-   8) Centrifuge the tubes at approximately 500 g for 5-10 minutes.    Aspirate all wash buffer (B) above gel surface.-   9) Add 180 μl lysis buffer (C) to each tube, add stopper and    incubate for 30 min on an orbital shaker at room temperature.-   10) Centrifuge the tubes at approximately 500 g for 5-10 minutes.

The RT activity recovered in the supernatant from step 10 can bequantitated with a sensitive RT activity assay, e.g. the Cavidi®HS-kits,which are based on the method described by Ekstrand et al 1996.

II) Protocol for Plasma Separation on Mini Columns

-   1) Label the desired amount of 15 ml plastic mini columns to    identify the samples to be analyzed.-   2) Prepare the wash buffer (B), 20 ml buffer/sample is needed-   3) Suspend the separation gel carefully into a 1:1 gel slurry and    transfer 1000 μl gel slurry to each column.-   4) The plasma to be analyzed is diluted 1:1 in binding buffer (A).    The system is optimized for the analysis of 500 μl plasma.-   5) Open the column at both ends and let the buffer in each column    run through. Add gently a maximum of 1000 μl sample according to 4)    on top of each column. Close the column before it run dry and let    the sample adsorb for 60 min before starting the wash procedure.-   6) Each column is washed with 2×10 ml wash buffer (B).-   7) When all wash buffer had passed through the columns add 200 μl    lysis buffer (C) to each column, let the column run dry and incubate    for 30 min at room temperature.-   8) Move the columns to a rack with sample collection tubes and add    another 300 μl lysis buffer (C) to each column. Collect the eluates    which contain the RT from the virions present in the original    samples.

The RT activity recovered in the the eluates from step 8 can bequantitated with a sensitive RT activity assay, e.g. the Cavidi®HS-kits, which are based on the method described by Ekstrand et al 1996.

Materials Used in a Preferred Embodiment

Separation gel: e.g. Fractogel® EMD TMAE, or Fractogel® EMD DMAEequilibrated in 150 mM MES (2-(N-Morpholino)ethanesulfonic acid) pH 6.0.Mini columns e.g. Biorad Poly-Prep® (7311553)

Plastic tubes

A) Binding buffer: 300 mM MES pH 6.0, 400 mM Potassium iodide (KI)

B) Wash buffer: 10 mM MES pH 6.0, 500 mM Potassium acetate (KAc)

C) Lysis buffer: A RT assay compatible buffer including a detergent e.g.1% Triton X-100 and a buffering substance e.g. 100 mM(N-(2-Hydroxyethylpiperazine-N′-(2-ethanesulfonic acid) (Hepes) pH 7.6.

SHORT DESCRIPTION OF THE DRAWINGS

FIG. 1 shows two diagrams A and B, which show the effects of addition ofdifferent cations to the RT reaction mixture at standard assayconditions.

Symbols: Ac⁻ (♦), Cl⁻ (▪), Br⁻ (▴), I⁻ (◯), SCN⁻ (⋄).

FIG. 2 is a diagram which shows the relation between amount of HIV RTrecovered according to the invention and amount of viral RNA measuredwith HIV 1 RNA PCR.

Symbols: pooled plasma from 100 healthy blood donors ♦, plasma from HIVinfected individuals ●

FIG. 3 is a diagram which shows detection of minute amounts ofretrovirus by concentrating the RT activity from a large volume of cellculture media

Symbols: 1% serum (open bar), 10% serum (closed bar).

EXAMPLES Example 1

Screening of the capacity of various chromatography media to immobilizeretrovirus (Table 1). 500 μl of a FIV spiked human plasma diluted 1:1 inbinding buffer was mixed with 100 μl of indicated gel. After one hour450 μl supernatant was withdrawn from each sample and replaced with 450μl lysis buffer. The samples were then incubated for another 30 minutesand the amount of RT activity in each supernatant and lysis fraction wasdetermined and calculated as a percentage of the RT activity added tothe original sample. There was a considerable variation in virus bindingcapacity among the media tested. The highest binding capacity was foundamong anionic ion exchange media with tertiary or quaternary amines. Therecovery of RT enzyme in the lysis fraction using the media with goodbinding capacity ranged from 53 to 100%, which indicated that no bindingto the anionic exchanger of the released RT occurred during virus lysis.Our main criteria for selection of separation media was good virusbinding capacity and high recovery of FIV RT.

Example 2

Recovery of RT activity from different retroviruses added to humanplasma (Table 2). Four samples of a human plasma were spiked with oneeach of four different retroviruses. The samples were diluted 1:1 insample dilution buffer and processed according to “Protocol for plasmaseparation on mini columns” using 500 μl of Fractogel TMAE highcap gel.Each RT was recovered in 300 μl lysis buffer and the amount of RT and Igin the lysates were determined and recalculated as a percentage of thecorresponding amounts in the original sample. Between 39 and 74% of theRT activity present in the original virus preparations were recovered inthe 300 μl lysate fraction. The reduction in Ig concentration wasbetween 2000 and 12000 times for the different samples.

Example 3

Effects of addition of different cations to the RT reaction mixture(FIG. 1). Chaotropic cations giving the indicated final concentrationswere added to Cavidi HS kit Lenti RT reaction solutions. The effect offive different cations on the activity of two RTs was evaluated: A) FIVvirions corresponding to 0.4 μU RT activity, B) Recombinant HIV 1 RTcorresponding to 0.6 μU RT activity. The RT activity at each cationconcentration was recalculated into percent of a control measured atstandard conditions. The inhibitory effect of different cations wasrelated to the chaotropic effect of the ions. The most chaotropiccations, SCN⁻ and I⁻ were inhibitory at all concentrations tested.Intermediate ions, Br⁻ and Cl⁻ stimulated the retroviral RTs atconcentrations below 100 mM but were increasingly inhibitory at higherconcentrations. From FIG. 1 it can be concluded that Cl⁻ or Br⁻ ions atconcentrations up to 70 mM and Ac⁻ ions up to 200 mM can be included inthe reaction mixture without causing adverse effects.

Example 4

Relation between amount of HIV RT recovered according to the inventionand amount of viral RNA measured with HIV 1 RNA PCR (FIG. 2). 200 μlsamples of EDTA plasma from HIV infected individuals, and from a poolobtained from 100 healthy blood donors, were processed according to“Protocol for batch separation of plasma”. The amount of RT activityrecovered from each sample was determined in an overnight RT assay usingCavidi HS kit Lenti RT. The RT activities obtained were recalculatedinto pg HIV 1 RT according to an internal standard curve. The amount ofHIV 1 RNA in each sample was measured by standard HIV 1 RNA PCR (Roche,Cobas Amplicor HIV monitor version 1.5). Values<50 RNA copies/ml havebeen plotted equal to 25 (FIG. 2). A strong correlation was foundbetween amount of plasma RT recovered according to the invention andamount of HIV RNA measured with PCR (r=0.85, p<0.001, Spearmancorrelation by ranks).

Example 5 Detection of Minute Amounts of Retrovirus by Concentrating theRT Activity from a Large Volume of Cell Culture Media (FIG. 3)

Eagles minimum essential medium (MEM) containing either 1% (open bar) or10% (filled bar) newborn calf serum (Gibco) was prepared. A small amountof FIV virions corresponding to 0.6 mU RT activity was added to 1, 5 and25 ml samples from each MEM/serum mixture. Each sample was passedthrough a 1 ml DEAE A25 Sephadex mini column, followed by a wash with 20ml wash buffer. Finally the RT activity was eluted into 300 μl elutionbuffer according to “protocol for plasma separation on mini columns”.With this procedure we recovered more than 70% of the virus associatedRT from 25 ml media as long as the serum concentration did not exceed1%. The corresponding figure at 10% serum was approximately 50% (FIG.3).

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TABLE 1 Screening of the capacity of various chromatography media toimmobilize retrovirus. *RT activity in *RT activity recov- *Total RTactivity Trade Name Type of interaction Active group Matrix supernatant(%) ered from gel (%) recovered (%) Fraktogel, DEAE anionic, weakR-CH₂N(C₂H₅)₂ methacrylate 6 68 74 DEAE-Si500 anionic, weakR-CH₂N(C₂H₅)₂ silica 9 96 105 Fraktogel, DMAE anionic, weak R-CH₂N(CH₃)₂methacrylate 9 73 83 Fraktogel, TMAE anionic, strong R-CH₂N + (CH₃)₃methacrylate 11 73 85 Fraktogel, TMAE anionic, strong R-CH₂N + (CH₃)₃methacrylate 12 100 112 high cap DEAE Ceramics anionic, weakR-CH₂N(C₂H₅)₂ ceramics 19 98 117 Hyper DF Macro prep High anionic,strong R-CH₂N + (CH₃)₃ methacrylate 21 72 93 Q Macro prep anionic, weakR-CH₂N(C₂H₅)₂ methacrylate 38 47 85 DEAE support Toyopearl DEAE anionic,weak R-CH₂N(C₂H₅)₂ methacrylate 40 64 104 650M AG4 - X4 anionic, weakR-CH₂N(CH₃)₂ acrylic 40 64 104 polymer QAE sephadex anionic, strongR-CH₂N + (C₂H₃OHCH₃)(C₂H₅)₂ agarose 42 53 96 A25 DEAE Sephadex anionic,weak R-CH₂N(C₂H₅)₂ dextran 46 56 102 A25 Express ion anionic, weakR-CH₂N(C₂H₅)₂ cellulose 50 57 107 exchange Sephadex G25 control nonedextran 81 38 118 *percent of the RT activity added to the originalsample.

TABLE 2 Recovery of RT activity from different retroviruses added tohuman plasma. RT added to plasma sample Virus (mU RT activity) RTRecovery (%) Ig recovery (%) FIV^(a) 0.24 66 0.023 BLV^(b) 0.68 41 0.014JSRV^(c) 0.10 74 0.047 MuLV^(d) 0.35 39 0.007 ^(a)Felineimmunodeficiency virus ^(b)Bovine leukemia virus ^(c)Jaagsiekte sheepretrovirus ^(d)Murine leukemia virus

1. Method of concentrating and recovering a reverse transcriptase (RT)enzyme activity from enveloped retroviruses present in a biologicalsample comprising the steps of contacting the biological sample in afirst buffer solution at a concentration in range of 100-500 mM ofbuffering substance and having a pH of 4.0-8.5 and optionally containingchaothropic ions at an ionic strength of up to 2 M with a virus-bindingmatrix, to attach virus particles present in the sample to the matrix,washing the matrix carrying the virus particles with a second buffersolution at a concentration of 1-100 mM of buffering substance andcontaining cations at a concentration of 0.1-1 M and having a pH of 4-9,to remove components interfering with viral enzyme activity, lysing theimmobilized virus particles in a third buffer solution at aconcentration of 10-500 mM of buffering substance and containing a nondenaturing detergent and having a pH of 4-9 which prevents the enzymefrom binding to the virus-binding matrix, and recovering theconcentrated viral enzyme activity from the third buffer solution. 2.Method according to claim 1, wherein the virus-binding matrix is ananion exchanger matrix.
 3. Method according to claim 2, wherein theanion exchanger matrix contains tertiary and/or quaternary amine groups.4. Method according to claim 1, wherein the first buffer is selectedfrom the group consisting of 150 mM MES pH 6.0, 200 mM Potassium iodide(KI).
 5. Method according to claim 1, wherein the second buffer isselected from the group consisting of 10 mM MES pH 6.0, 500 mM Potassiumacetate (KAc).
 6. Method according to claim 1, wherein the third bufferis selected from the group consisting of enzyme assay compatible buffersincluding a detergent and a buffering substance.
 7. Method according toclaim 1, wherein the biological sample is selected from the groupconsisting of serum and plasma samples.
 8. Commercial package containingwritten and/or data carrier instructions for performing laboratory stepsfor concentration and recovery of a reverse transcriptase enzymeactivity from enveloped retroviruses present in a biological sample, andthe components a virus-binding matrix, a first buffer solution at aconcentration in range of 100-500 mM of buffering substance and having apH of 4.0-8.5 and containing chaothropic ions at an ionic strength of upto 2 M, a second buffer solution at a concentration of 1-100 mM ofbuffering substance and containing cations at a concentration of 0.1-1 Mand having a pH of 4-9, a third buffer solution at a concentration of10-500 mM of buffering substance and containing a non denaturingdetergent and having a pH of 4-9, and optionally Mini columns, andPlastic tubes.
 9. Commercial package according to claim 8, wherein thevirus-binding matrix is an anion exchanger matrix, the first buffer isselected from the group consisting of 150 mM MES pH 6.0, 200 mMPotassium iodide (KI), the second buffer is selected from the groupconsisting of 10 mM MES pH 6.0, 500 mM Potassium acetate (KAc), thethird buffer is selected from the group consisting of enzyme assaycompatible buffers including a detergent and a buffering substance. 10.Commercial package according to claim 9, wherein the anion exchangermatrix contains tertiary and/or quaternary amine groups.
 11. Methodaccording to claim 2, wherein the first buffer is selected therefor thegroup consisting of 150 mM MES pH 6.0, 200 mM Potassium iodide (KI); thesecond buffer is selected from the group consisting of 10 mM MES pH 6.0,500 mM Potassium acetate (KAc); and wherein the third buffer is selectedfrom the group consisting of enzyme assay compatible buffers including adetergent and a buffering substance.
 12. Method according to claim 11,wherein the biological sample is selected from the group consisting ofserum and plasma samples.
 13. Method according to claim 12 wherein theRT activity is quantitated with a sensitive assay.